2281

711 ANDROGEN METABOLISM IN THE RHESUS MONKEY Y. Yamamoto, A. Manyon, R.Y. Kirdani and A.A. Sandberg Roswell Park Memorial Institute, Buffalo, New York 14263

Received 2-10-78 ABSTRACT A mixture of 3H-testosterone (T) and 1~C-4-androstene-3,17-dione (A) was injected intravenously into 2 (I and 117 rhesus monkeys (Macaca mulatta). A third monkey (III) was injected with ~ - T only. Urine and bile, samples were collected at intervals for 6 hours following the injection. The excretion, conjugation and aglycone metabolites of the steroids injected were studied using these samples. Of the injected dose, animal I (male) excreted 32% ~ and 23% I~C in the bile and 30% 3H and 21% I~C in the urine in 6 hours. Animal II (female), however, had a comparatively higher biliary excretion (66% ~, 40% I~C), but a urinary excretion (18% ~, 13% I~C) comparable to that of animals I and III. The averages in the bile of the 3 animals were: unconjugated compounds 3%, glucoslduronates 78%, sulfates 9%, sulfoglucosiduronates 5% and disulfates 3%; and in urine, 5% unconjugated, 92% glucoslduronates and 3% sulfates. The aglycones obtained following hydrolysis were separated by chromatography on Lipidex 5000, further purified by thin layer and paper chromatography and identified by co-crystallization. The major matabolites from 3H-T were androsterone and 58-androstane-3~,178-dlol, whereas that from I~C-A was androsterone. Other metabolltes identified were: etiocholanolone (3~-hydroxy-58-androstan-17-one); T, epitestosterone (epi-T), (17~-hydroxy-4-androsten-3-one); epiandrosterone (38hydroxy-5~-androstan-17-one) and 5~-androstane-3~,178-diol. The results indicate that while androgen metabolism in the rhesus monkey is similar to that of the baboon and human in conjugate and metabollte formation, the rate of excretion was significantly different, resembling more closely that of the baboon than the human.

INTRODUCTION Primates are regarded as suitable surrogates for humans in many fields of research.

In work on steroid hormones, Setchell et al.

(i)

reported on the urinary steroid excretion and conjugation in baboons and showed that this animal is a suitable model for the study of corticosteroid metabolism.

Volume 31, Number 5

In a series of studies on androgen metabolism in

~

~

~

~

~ x ~

~

May, 1978

different animal species, we have previously reported data on dogs and baboons [2-4], indicating that the latter closely resembled the human with respect to androgen metabolism.

Reports

[5-8] on the endogenous levels of

steroids in urine and plasma of rhesus monkeys have been published, and the data suggest a similarity of androgen metabolism with that in the human.

However, a study of androgen metabolism in the rhesus monkey has

not been reported as yet. The above circumstances prompted us to study androgen metabolism in the rhesus monkey by injecting separately or admixed

~ - T and I ~C-A.

Following the injection, urine and bile were collected, and the urinary and biliary excretion, conjugation and metabolite formation defined.

The

results are presented and discussed comparatively with those observed in the human and baboon.

MATERIALS AND METHODS The injected radioactive steroids, T - I B , 2 ~ - ~ (S.A. = 139mCi/mg; 40.3 Ci/mmole); A -4-1~C (S.A. = 0.2mCi/mg;57.6mCi/m~ole)were purchased from the New England Nulcear Co., Boston, Mass.; their purity was checked by thin layer chromatography before use. Non-radioactive steroids used as standards for thin layer and paper chromatography and as carriers for cocrystallizations were purchased from Steraloids, Inc., Wilton, N.H. Three animals were used, a 4kg male (I), a 9kg female (II) and a 6kg female (III) rhesus monkeys (Macaca mulatta). A mixture of ~H-T (60~Ci) and I~C-A (20~Ci) was injected intravenously into animals I and II. Animal III received 3H-T (60~Ci) only£ Under intravenous anesthesia and sterile conditions, the bile duct was cannulated and the urinary bladder catheterized directly through an abdominal incision. Urine and bile were collected at intervals for a total of 6 hours following the injection. Bile and urine samples were eluted from DEAE-Sephadex A-25 columns (Pharmacia Fine Chemicals, Inc., Piscataway, N.J.); the system used for all fractionations contained 3 columns (K9/60,0.9cmx60cm; K9/30,O.9cmx30cm; K9/15,0.9cmxl5cm). The columns were packed with Sephadex A-25 equilibrated with water, and the effluent from one column was applied directly to the origin of the next through 0.3cm polyethylene tubing; this system afforded superior resolution as compared to the use of one long column. Distilled water was the initial eluent followed by a 0-0.6M NaCI linear gradient and a 2.0M NaCI wash.

T

~m " ~ o

X ~ m

713

Fractions containing the glucuronide conjugates were combined and aliquots hydrolyzed with beef liver B-glucuronidase (Sigma Chemical Co., St. Louis, Mo.) in O.IM acetate buffer, pH 5. 500-1000 Units of enzyme/ml of buffer were used. Two other incubations, one without enzyme and one containing saccharo-l,4-1actone (Calbiochem Co., San Diego, Calif.; 30mg/ 15ml) were also used. Mono- and di-sulfate conjugate fractions were hydrolyzed by solvolysis procedures as described by Bursteln and Lieberman [9]. Sulfo-glucuronide dlconjugates were sequentially hydrolyzed (B-glucu ronidase hydrolysis followed by solvolysls). Aglycones obtained from ~-glucuronidase hydrolysis were separated into metabolite groups by column chromatography in Lipidex 5000 (Packard Instrument Co., Inc., Downer's Grove, Iii.). Before use, the Lipldex was equilibrated with hexane; two 1.0cm diameter columns (Pyrex No. 2145 and No. 7282) were packed to a height of 17cm and 20cm and connected. Metabolites were eluted with 500ml of purified hexane followed by a linear gradient mixing 400ml of hexane with 400ml of 20% benzene in hexane. The column was washed with a polar solvent (hexane:chloroform:methanol; 70: 20:10) to elute polar metabolites. Metabolites obtained from Lipidex columns and aglycones from solvolysis were further purified by thin layer chromatography on silica gel GF in a chloroform:acetone (36:4) system and by paper chromatography on Whatman filter paper No. i in a hexane:methanol:water (10:9:1) system. Radioactivity was determined in both thin layer and paper chromatography in a Packard scanner model 7201. Final identification of metabolites was achieved by co-crystallization to constant specific activity. Liquid scintillation counting was performed in a Packard tri-carb spectrometer model 2450. Radioactive allquots were added to 10ml aqueous counting sclntillant, ACS, purchased from the Amersham Corp., which had been diluted with toluene (3:1) for economy. Determination of radioactivity in the highly quenched bile samples was achieved by dilution of the bile to 5ml with distilled water followed by combustion of an aliquot of this mixture in a Packard sample oxidizer model 306.

RESULTS i.

Excretion:

Excretion of radioactivity in bile and urine of the

3 monkeys following intravenous injection of SH-T and I~C-A is given in Table i.

Animal I excreted 32% SH and 23% I~C in the bile and 26% SH and

21% I~C in the urine during 6 hours of collection.

Animal III, into

which only SH-T was injected, excreted 34% in the bile and 33% in the urine.

Thus, animals I and III showed similar excretion patterns and

rates.

However, animal II excreted radioactivity preponderantly in the

bile:

66%

~

and 40% I~C compared to the urine which contained 18%

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and 13% I~C.

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The recovery of 3H was always higher than that of I~C, both

in bile and urine. 2.

Conjugates:

To fractionate the conjugates, bile and urine were

directly applied to a DEAE-Sephadex A-25 column and eluted as described above.

The elution pattern of bile is shown in Fig. i; 6 conjugate

fractions (F-I to F-6) were obtained.

3 H 14C (xlO3 DPM) 40

-F-2-

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~ 20

5

B

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'2.0M NoCI

......s...I ....~....~ s

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20



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0.6M NoCI Lineor G¢odient

-4

120

I 140

o I 160

TUBE NUMBER

Fig. i. DEAE-Sephadex A-25 column chromatographic separation of conjugates in rhesus monkey bile. Uncharged fraction (F-l) was eluted with water; the monoglucosiduronates (F-2), the monosulfates (F-3) and the sulfo-glucosiduronates (F-4) with a 0.6M NaCI - water linear gradient. A 2.0M NaCI wash eluted undetermined conjugates (F-5) and disulfates (F-6). 3H = solid line; I~C = dotted line.

The first fraction (F-l) was eluted with distilled water and contained uncharged compounds.

About 26% of the compounds of this fraction

were unconjugated, since they were extractable in ether:

ethyl acetate

(i:i).

Fractions F-2 to F-4 were eluted with a 0-0.6M NaCI linear gra-

dient.

F-2 was comprised of 2 peaks; this fraction was hydrolyzed by B-

glucuronidase as shown in the following:

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]Dm

I WC

3H

96.4% 3.8% 3.8%

Hydrolysis Control Inhibition F-3 was comprised of 3 peaks.

97.6% 5.3% 4.1%

Each was individually collected and hy-

drolyzed by solvolytic procedures.

The results are shown in Table II.

Thus, this fraction was tentatively considered to consist of sulfates, since they were all solvolyzed.

F-4 also contained 3 peaks, each of

which was sequentially hydrolyzed (Table II) and determined to be sulfoglucoslduronate conjugates.

F-5 was eluted at the beginning of the 2.0M

NaCI wash and was not successfully hydrolyzed either enzymatically or by solvolytic procedures or both; this fraction remained undetermined.

F-6

was eluted out in highly ionic NaCI wash and solvolyzed (Table II).

Thus,

the 6 fractions obtained were:

F-I = uncharged; F-2 = glucosiduronates;

F-3 = sulfates; F-4 = sulfoglucosiduronates; F-5 = undetermined and F-6 = disulfates. The elution pattern of urine in DEAE-Sephadex A-25 is shown in Fig. 2; there were no diconjugates excreted into the urine.

The uncharged

fraction, F-l, was eluted with distilled water and 43% of it was unconjugated (extractable into ether:ethyl acetate; i:i). ted in the O-0.6M NaCI linear gradient. of 2 peaks.

F-2 and F-3 were elu-

F-2, as in bile, was comprised

This fraction was hydrolyzed by 8-glucuronidase as follows:

Hydrolysis Control Inhibition

3H 82.2% 12.3% 22.1%

I~ C 89.1% 14.5% 19.5%

F-3 was comprised of 2 peaks which were combined and solvolyzed, with 76% of the ~

and 78% of the I~C becoming extractable after solvolysis.

Therefore, 3 fractions were obtained from urine: glucoslduronates and F-3 = sulfates.

F-I = uncharged; F-2 =

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..,,.4

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TUBE NUMER

Flg. 2. DEAE-Sephadex A-25 column chromatographic separation of androgen conjugates in rhesus monkey urine. Distilled water eluted the uncharged fraction (F-l). A 0.6M NaCI - distilled water linear gradient eluted the monoglucosiduronates (F-2) and monosulfates (F-3). The 2.0M NaCI wash did not elute any additional conjugates. The conjugate patterns in bile were studied as a function of tlme and the results are shown in Table III and Fig. 3.

The percent distri-

bution of conjugates in the bile at each time period (Fig. 3) showed that glucoslduronate formation decreased gradually wlth time (95% to 80%); on the other hand, sulfates and diconjugates were formed in increasing amounts, even though their percentages were small.

In the urine, the de-

crease of glucosiduronates and the increase of sulfates were not as obvious as those in the bile, and more than 90% of the radioactivity was consistently recovered as glucosiduronates.

Table III shows that the con-

Jugates in bile and urine were mostly comprised of glucosiduronates.

Fur-

thermore, conjugate formation was particularly extensive during the initial 2 hours after injection; it is expressed as the percent dose conjugated per minute in Fig. 4.

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m

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URfNE I00 m %

i0-60 60-120120-240 240-360 TIME Iminutes) m m

m

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0 TIME(minufu)

Fig. 3. Percent distribution with time (following injection of radioactivity (3H) in the conjugate fractions of rhesus monkey II bile and urine. Diconjugates include sulfo-glucosiduronates, disulfates and undetermined conjugates (F-V). Open bars = glucoslduronates, dark bars = sulfates, and hatched bars = dlconjugates.

3.

Metabolltes:

The unconjugated metabolltes

in bile and urine, ex-

tracted from uncharged fraction F-I with ether:ethyl acetate (i:i), were separated by thin layer chromatography.

Two peaks were obtained corre-

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I.

0

x z)m

721

P m c ~ Of /~lmini~m~l Dose ~10"3 %)

Percent Of Administered Dose (xlO - 3 % )

400 -

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i 0-30

30-60 60-120120-240 240-360 T I M E (mintde)

Fig. 4. Conjugate excretion expressed as percentage of administered dose per minute in each collection period. The conjugate fractions of ~ T in rhesus monkey II bile have been used as examples. As in Fig. 3, open bars = glucosiduronates, dark bars = sulfates and hatched bars = dlconjugates. sponding with standard A and polar metabolities,

respectively.

Further

analysis of this fraction was not carried out. The glucoslduronate fractions (F-2) from bile and urine after Bglucuronldase hydrolysis were further separated by Lipidex 5000 column chromatography.

The separation pattern for bile is shown in Fig. 5.

Hexane (in the first 500ml) eluted monohydroxy metabolites, P-4.

peaks P-I to

A hexane - 20% benzene:hexane linear gradient eluted dihydroxy me-

tabolites

(peaks P-5 to P-7).

The polar solvent wash (hexane:chloroform:

methanol;

70:20:10) eluted highly polar metabolites

(peak P-8).

The

separation pattern for urine shown in Fig. 6 was similar to that of bile, and peaks P-I to P-8 were obtained.

722

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T

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~ , o ~" ]Din

~c I

~

(xlO3 DPM)/

I000

P'e~___H~.:CNo.:~ (70:20=10) P-2 75

P-6

20% Ehmz.Ha.H~. Lbleor

15

Grodlent ~-7

0

20

40

60

80 I00 TUBE NUMBER

120

NB~

140

160

Fig. 5. Lipidex 5000 column chromatographic elution pattern of androgens in glucosiduronate fraction of rhesus monkey bile. Hexane eluted monohydroxy metabolites (P-I artifacts; P-2 androsterone; P-3 ~tlocholanolone; P-4 T and epi-T). A 20% benzene in hexane and hexane linear gradient eluted dihydroxy metabolites (P-5 5~-androstane-3~,17~-diol; P-6 5B-androstane-3~,17B-diol; P-7 unidentified) and polar solvents (hexane: chloroform:methanol; 70:20:10) eluted highly polar metabolites (P-8). The agylcones separated by Lipidex column chromatography were further purified either on thin layer or paper chromatography and finally identified by co-crystallization.

P-I was less polar than the monohydroxy me-

tabolltes and regarded as an artifact; no further analysis was performed. P-2 was obtained as a sole peak on paper chromatography coincidently with standard androsterone and identified by co-crystalllzation as such. was identified as etiocholanolone.

P-4 was separated into 2 peaks on

paper, which were identified as T and epi-T.

P-5 and P-6 were eluted on

thin layer with Rf's similar to those of standard androstanediols: androstane-3~,17B-diol

5~-

and 5~-androstane-3~,17~-diol were identified in

P-5 and 5B-androstane-3~,17B-diol than 5B-androstane-3~,17B-diol not identified.

P-3

in P-6.

P-7 was slightly more polar

on thin layer, but the metabolites were

P-8, polar metabolites, was separated on thin layer in

~

-~m . l . o

z ~D ,,,,

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SH 14C )

~K~ DPM~l

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4 --

P-2p_3

....

P-6

A_J

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I - b L :Chlo: M e O H (70:20:100)

20% Be~.:Hex. - H e L Liemar Grodkmt

25 P-5 o

2o

40

eo

,o

P-7 ,oo

~

,40

i 160

TUBE NUMBER

Fig. 6. Lipidex 5000 column chromatographic elution pattern of androgen metabolites in glucosiduronate fraction of rhesus monkey urine. Hexane eluted monohydroxy metabolites (P-i artifacts; P-2 androsterone; P-3 etiocholanolone; P-4 T and epi-T). Linear gradient system eluted dlhydroxy metabolites (P-5 5~-androstane-3~,178, diol; P-6 58-androstane3e,178,diol; P-7 unidentified) and polar solvents (hexane:chloroform: methanol; 70:20:10) eluted highly polar metabolites (P-8) which were not identified. system chloroform:ethanol

(9:1), and 2 peaks were obtained, one corre-

sponded with standard 5~-androstane-3~,16~,17B-triol

and the other was

more polar than this trlol. The aglycones solvolyzed from the sulfate fraction of bile were separated on thin layer and 3 major peaks were obtained.

Epiandrosterone and

5~-androstane-3~,17B-diol were identified by co-crystallization.

One of

the aglycone metabolites in the disulfate fraction was identified as 5~androstane-3~,17~-dlol.

The major metabolites in the sulfo-glucosiduronate

fraction were polar compounds which were not further identified. The metabolites identified in the glucosiduronate fraction of urine and bile were quantitated and the results expressed as percent of the administered dose in Tables IV (animal I) and V (animal II).

The major me-

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tabolites (total urine + bile) were androsterone, etiocholanolone and 5~androstane-3~,17B-diol.

In the 17-keto steroids category, in animal I,

androsterone formation was higher than that of etiocholanolone.

In animal

II, however, etlocholanolone formation was extensive especially from SHlabeled precursor.

More etiocholanolone was excreted in bile than in

urine, except in animal II.

The SH/I~C ratio in the 17-keto steroids was

smaller than the injected ratio.

Even though T and epi-T were identified

as glucosiduronates, their formation was small (0.1-1.2%).

T-glucosi-

duronate was formed from SH-T predominately (isotope ratio = 3.25 for animals I and II), whereas epi-T was formed from I~C-A (isotope ratio ffi 1.9 and 4.8 in animals I and II, respectively).

In the dihydroxy category,

more 5~-androstane-3~,17B-dlol and 5B-androstane-3~,17B-dlol were'converted from ~ - T than from I~C-A.

This is indicated by a higher

~/I~C

ratio than the injected one. 5~-Androstane-3~,17~-diol was the predominant metabolite (19%) of SH-T and excreted largely into bile.

5~-Androstane-

3~,17~-dioi was excreted in a very small percentage with about the same isotope ratio as that of the injected dose.

Unidentified metabolites to-

taled 17-27% of the injected dose.

DISCUSSION Metabolltes and conjugates of androgens are excreted preponderantly into the bile in lower mammals, such as the rat [i0|, cat [II], rabbit [12] and dog [2,3], whereas the human and baboon excrete mainly in the urine [13,14].

The excretion observed in rhesus monkeys situates this

animal intermediately between these 2 groups.

However, animal III showed

preponderant excretion in bile and the pattern is similar to that of lower animals.

Another interesting feature in the rhesus monkey is that metabo-

lites of A were recovered in much lower amounts than those of T, as com-

~

.u

~.~

I

727

:D~

pared to the almost equal excretion of these metabolites in the human and baboon [4]. The conjugate patterns of rhesus monkey samples showed close resemblance to those of the baboon and human [4], i.e., the Lipidex column separation pattern of rhesus monkey bile was very similar to that of baboon, and that of the urine to those of the baboon and human.

Gluco-

siduronates were the major conjugates and constituted approximately 85% of the total recovery.

Sulfates were the second largest fraction, but

constituted only 6-9%.

The excretion of these 2 conjugates was observed

in 30 to 120 minutes after the injection.

Diconjugates,

sulfoglucosidu-

ronates and d~sulfates were found only in the bile, though in small quantity (4-5%).

Endogenous sulfates of 17-keto steroids have been reported

in the rhesus monkey [6,7]; diconjugates, however, were not well established.

The present study tentatively proved that sulfoglucosiduronates

and disulfates are formed by the rhesus monkey. The major aglycone metabolites in the glucosiduronate fraction were two 17-keto steroids,

i.e., androsterone and etiocholanolone,

5B-androstane-3~,17~-diol.

and a diol,

In the urine of monkey I (male) androsterone

was excreted in amounts higher (c__aa.4-fold) than those of etiocholanolone; the androsterone/etiocholanolone monkey II (female) urine.

ratio, however, was about 2 in

This is in agreement with the report that the

male rhesus monkey excreted 5-7 times more androsterone than etiocholanolone, whereas they are excreted almost equally in the female [6].

Even

though we only analyzed samples from one male and one female, the ratio of the major labeled 17-ketosteroids was similar to the endogenous one in the rhesus monkey (5-8), but higher than that in the human (17).

728

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T

I

lw, O

z

~11

The excretion of 17-keto steroids was significantly different in monkey bile and urine, i.e., etiocholanolone was found in larger quantity than androsterone.

In baboon, etiocholanolone was excreted in

small quantities, in both urine and bile, compared with androsterone

[4]. Both T and epi-T were isolated as glucosiduronates; the latter was mostly derived from A.

These 2 substances were also excreted in the

baboon and human [4], though in much smaller amounts than in the rhesus monkey. In the rhesus monkeys 58-androstane-3a,178-diol was the most significant metabolite derived from T, the amounts being 4-5 times those derived from A.

The high 3H/I~C ratio suggests that T-glucosiduronate "

serves in the pathway for the conversion to 58-diol, as reported for the human [15], rat [12] and dog [2,3].

The baboon also excreted 58-diols

as the major metabolites, whereas its formation was relatively small in the human [4,15]. Two other diols were excreted, i.e., 5a-androstane-3~,178-diol and 5~-androstane-3a,17~-diol, with the former being excreted 15-25 times more than that of the latter.

It is of interest that the formation of

17a-diol exceeds 178-diol in the baboon [4]. In the sulfate fractions of bile, epi-androsterone and 5~-androstane3~,178-dioi were identified, the latter being found as the disulfate. Epi-androsterone has been identified in baboon bile [4] as a sulfate and 5~-androstane-3e,178-diol in the human [16] and rat [lO] bile as a diconjugate. The overall results indicate that the rhesus monkey excretes androgen metabolites with quantitative differences from the baboon and human,

~

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"r ~

m

729

but that the conjugation patterns were quite similar among these 3 primates.

The aglycone metabolites are more similar to those of the

baboon than of the human.

ACKNOWLEDGMENTS This study has been supported in part by grant AM-01240 from the National Institutes of Health. We wish to thank Mrs. Diane Smith, Mrs. Cathy Russin and Miss Anne Marie Contl for their technical and clerical assistance.

REFERENCES i.

Setchell, K.D.R., Axelson, M., Simarina, A.I., and Gontscharow, N.P. (1976). Yamamoto, Y., Osawa, Y., Kirdani, R.Y. and Sandberg, A.A. J. STEROID BIOCHEM. In press (1978). Yamamoto, Y., Osawa, Y., Kirdani, R.Y. and Sandberg, A.A. STEROIDS In press (1978). Yamamoto, Y., Osawa, Y., Kirdani, R.Y. and Sandberg, A.A. J. STEROID BIOCHEM. In press (1978). Tolson, W.W., Johnson, T.R. and Mason, J.W. J. LAB. CL. MED. 68, 981 (1966). O'Malley, B.W. and Lipsett, M.B. STEROIDS 8, 711-718 (1966). Pal, S.B. ACTA ENDOCRINOL. 67, 711-720 (19~i). Aso, T., Goncharov, N., Cekam, Z. and Dicfalusy, E. ACTA ENDOCRINOL. 82, 644-651 (1976). Burnstein, S. and Lieberman, S. J. BIOL. CHEM. 233, 331-335 (1958). Matsui, M., Kinuyama, Y. and Hakozaki, M. STEROIDS 24, 557-573 (1974). Archer, S.E.H., Scratcherd, T. and Taylor, W. BIOCHEM. J. 94, 778782 (1965). Taylor, W. and Scratcherd, T. BIOCHEM. J. 104, 250-253 (1967). Sandberg, A.A. and Slaunwhite, W.R., Jr. J. CL. INVEST. 35, 13311339 (1956). Hellman, L., Bradlow, H.L., Frazell, E.L. and Gallagher, T.F. J. CL. INVEST. 35, 1033-1044 (1956). Robel, P., Emiliozzi, R. and Baulieu, E.-E. J. BIOL. CHEM. 241, 20-29 (1966). Laatainen, T. and Vihko, R. STEROIDS 14, 119-131 (1969). Fieser, L.F. and Fieser, M. STEROIDS, Reinhold Publishing Corporation, New York, 1959, p. 524. J. STEROID BIOCHEM. ~, 809-816

2. 3. 4. 5. 6. 7. 8. 9. i0. Ii. 12. 13. 14. 15. 16. 17.

Androgen metabolism in the rhesus monkey.

2281 711 ANDROGEN METABOLISM IN THE RHESUS MONKEY Y. Yamamoto, A. Manyon, R.Y. Kirdani and A.A. Sandberg Roswell Park Memorial Institute, Buffalo, Ne...
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